Anti-Proliferative Effects of Palm Vegetation Liquor And Extracts Thereof In Preventing Pancreatic Cancer

ABSTRACT

Present invention relates to a composition to inhibit cancer cell proliferation, wherein said composition comprises oil palm extracts. The composition is useful for prevention of pancreatic cancer by inhibiting clonogenicity, inducing apoptosis, regulating gene expression, inducing anti-invasive effect, and induces cell cycle arrest in S phase. Present invention also discloses a composition that inhibits NF-κB activity and reduces cell invasion, cell migration and metastasis. Present invention further discloses the use of therapeutically effective amount of a composition in inhibiting the growth of pancreatic cancer.

FIELD OF THE INVENTION

The present invention relates to a composition of anti-proliferativeeffects in cancer. More particularly, relates to a palm-basedcomposition that includes but is not limited to phenolic compounds foruse in preventing pancreatic cancer.

BACKGROUND OF THE INVENTION

The oil palm fruit (Elaeis guineensis) contains large amounts of lipidor water soluble bioactive agents such as vitamin E (tocopherols,tocotrienols), carotenoids and phytosterols. The lipid-solubletocotrienols are shown to have anti-tumor effects on human cancer cellslike prostate, breast, colon, melanoma, and lung cancers. Water-solubleoil palm phenolics (OPP) have been demonstrated to show potentialanticancer properties. Sambanthamurthi et al (2011) showed thatphenolics inhibit tumour progression in tumour-inoculated mice; howeverthere is no disclosure on tumour growth inhibition in pancreatic cancerusing palm vegetation liquor. Sekaran et al (2010) have demonstratedthat OPP can inhibit proliferation and induce apoptosis in differentcancer cell lines such as breast, lung, and skin cancers in vitro. Itwas also found that OPP reduces tumor growth in vivo. However, there isno study available to disclose the effect of oil palm bioactive agentson pancreatic cancer cell growth. Therefore, it is an object of presentinvention to utilize the extract from this oil palm vegetation liquor intreating pancreatic cancer (PaCa).

Pancreatic cancer (PaCa) is one of the leading causes of cancer deathwith poor survival rate due to its aggressiveness and early metastases.Despite more focused research in this area recently, the mortality ratefor PaCa has remained fairly high. The common treatments for PaCapatients are pancreatectomy, or surgical resection of the organ,followed by chemo, targeted and/or radiotherapies.

Gemcitabine is a purine analog which is used as a standard clinicalchemotherapeutic agent for advanced PaCa treatment; however this agenthas shown a poor tumor response rate with short survival time. Erlotinibis a targeting epidermal growth factor receptor tyrosine kinase which isused as an adjuvant to gemcitabine by inhibiting cell growth signalingbut again showing marginally improved survival benefits in clinicaltrials.

Detrimental side effects of these chemotherapeutic agents availabletargeting PaCa such as heart attack, stroke and death have beendocumented. This results in limited advantage due to their dose limitingtoxicity to normal tissues. Thus, it is an object of present inventionto provide an alternative to enhance chemotherapeutic activity forpancreatic cancer with minimal adverse effects.

Another object of present invention is to curb the adverse effects andchemoresistance suffered by most gemcitabine-treated patients. Itbecomes crucial to develop a novel therapeutic agent in order to improveprognosis with natural plant extracts.

It is yet another object of present invention to provide a bioactivedietary agent used in combination with drug to achieve the pleiotropiceffects which lessen the toxicity and dose requirements of therapeuticsalone.

The nuclear factor κB (NF-κB) is widely used as a regulator of genesthat control cell proliferation and cell survival, and it playsimportant roles in cancer cell transformation, cell invasion, andapoptosis. P65 is a subunit of NF-κB that interacts with promotersequence of target genes which in turn induces the expression of genesinvolving in inflammatory, anti-apoptosis, and proliferation. Thissignaling pathway has been targeted as a strategy for anti-cancertherapy in PaCa patients. NF-κB is a common and constitutively expressedtranscription factor which is activated through a wide variety ofstimuli such as inflammation and oxidative stress. On stimulation, NF-κBis translocated to the nucleus and binds to the promoters of its targetgenes to begin transcription of specific genes. Overexpression of NF-κBmay results in apoptosis resistance, angiogenesis, migration andinvasion of cancer cells.

Studies have shown that NF-κB is constitutively activated inapproximately 67% of pancreatic adenocarcinomas compared to normalpancreatic tissue, and this is associated with aggressive stagepancreatic cancer and drug resistance. Based on the role of NF-κB inregulating carcinogenic activity in pancreatic cancer and theanti-inflammatory behavior of palm vegetation liquor, present inventionserves the purpose to describe the effect of palm vegetation liquor inpancreatic cancer cell model, characterized in the potentialdown-regulation of the NF-κB pathway.

Thus, it is yet another object of present invention to provide acomposition for anti-proliferation in pancreatic cancer via regulationof NF-κB pathway.

It is a further object of the present invention to provide an improvedcomposition and method based on compounds extracted from vegetationliquor i.e. aqueous stream of the palm oil milling process for providinginhibitory effect against pancreatic cancer.

Accordingly, present invention also relates to an extraction process ofbioactives from the oil palm vegetation liquor from the milling process.The processing of oil palm produces large amounts of vegetation liquorrich in phenolic compounds, shikimic acid, fruit acids, fruit sugars andglycerol which can be further enriched using conventional membranefiltration technology.

Further objects and advantages of the present invention may becomeapparent upon referring to the preferred embodiments of the presentinvention as shown in the accompanying drawings and as described in thefollowing description.

SUMMARY OF THE INVENTION

The invention relates to a composition to inhibit cancer cellproliferation, wherein said composition comprises oil palm phenolicsderived from oil palm extracts and vegetation liquor. The composition isuseful for prevention of pancreatic cancer from PANC-1 and BxPC-3 celllines by inhibiting clonogenicity, inducing apoptosis, regulating geneexpression, anti-invasive effect, and induces cell cycle arrest in Sphase.

The apoptosis is associated with inducing caspase, inhibiting cellsurvival proteins and inhibiting anti-apoptotic protein. Theanti-apoptotic protein disclosed herein is Survivin. The compositionalso down-regulates Bcl-XL gene expression which is from Bcl-2 family.

The composition induces apoptosis by increasing expression ofpro-apoptotic proteins such as cleaved caspase 3, caspase 9 or Poly(ADP-ribose) polymerase (PARP).

Present invention also discloses a composition that inhibits NF-κBactivity via down-regulating p65 subunit activity. Vascular endothelialgrowth factor (VEGF) and matrix metalloproteinase 9 (MMP9) geneexpressions are down-regulated to reduce cell invasion, cell migrationand metastasis. The composition may be provided as compounds withpharmaceutically acceptable carriers.

Present invention further discloses the use of therapeutically effectiveamount of a composition in the preparation of a medicament forpreventing or inhibiting the growth of pancreatic cancer in anindividual by administering to an individual in need thereof. Thecomposition is accompanied with or without conventional chemotherapy orradiation therapy or surgery, or it may be administered orally or as afood supplement.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings, in which:

FIG. 1 shows the effect of phenolic-rich extract from oil palmvegetation liquor designated palm juice (PJ) on pancreatic cancer cellline PANC-1 and BxPC-3 survival and growth (A and B) and clonogenicity(C and D);

FIG. 2 shows the apoptosis analysis by Histone Kit/DNA ELISA method (Aand B) and flow cytometry (C and D);

FIG. 3 shows the effect of PJ on gene expression;

FIG. 4 shows the response of PJ on cell cycle regulation in PANC-1 (A)and BxPC-3 (B) cancer cell lines;

FIG. 5 shows the down regulation of NF-κB activity following PJtreatment in PANC-1 and BxPC-3 cells; and

FIG. 6 shows the inhibition of cell invasion and migration in PANC-1 andBxPC-3 cell lines.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments herein and the various features and advantageous detailsthereof are explained more fully with reference to the non-limitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to notunnecessarily obscure the embodiments herein. The examples used hereinare intended merely to facilitate an understanding of ways in which theembodiments herein may be practiced and to further enable those of skillin the art to practice the embodiments herein. Accordingly, the examplesshould not be construed as limiting the scope of the embodiments herein.

Present invention provides palm juice (PJ) which constitutes a watersoluble phenolic-rich extract from the palm fruit (Elaeis guineensis)and/or its vegetation liquor and has documented high antioxidant,anti-inflammatory, and anti-carcinogenic activity in breast cancercells.

Present invention also discloses anti-tumor effects of the phenolic-richextract by treating two pancreatic cancer cell lines (PANC-1 and BxPC-3)with different doses of palm juice (PJ). PJ was observed to induceanti-proliferative, apoptotic and anti-invasive effects using the(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)(MTS) assay, cytoplasmic histone-DNA fragment quantification andmatrigel invasive assays in a dose dependent manner. Real-time PCR hasconfirmed the anti-invasive effects induced by PJ through a decrease inthe gene expressions of MMP-9 and VEGF. Flow cytometer is used todemonstrate that cells arrest in S phase in cell cycle analysis. Westernblot analysis has also been conducted to show that apoptosis induced byPJ was associated with down regulation of expression in Survivin andBcl-XL gene and up regulation of expression in cleaved caspase 3,caspase 9 and PARP gene. These results demonstrate the anti-tumoractivity of PJ in pancreatic cancer cells, providing initial evidencetowards its potential therapeutic uses.

BEST MODE FOR CARRYING OUT THE INVENTION

The method of preparation and use of the present invention is furtherillustrated by the following experimental examples. It should beunderstood that these experimental examples, while indicating preferredembodiments of the invention, are given by way for better elucidationonly. A person skilled in the art can ascertain the essentialcharacteristics and embodiments of this invention, therefore variouschanges may be provided to adapt to various usages and conditions.

Materials and Methods Cell Culture, Reagents and Antibodies:

Human PaCa cell lines, including PANC-1 and BxPC-3 were grown inDulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetalbovine serum (FBS) and 1% penicillin and streptomycin in 5% CO₂. Palmjuice (PJ) extracted from oil palm was prepared at a stock concentrationof 1500 mg/ml GE (gallic acid equivalents). Protease inhibitor cocktail,primary antibodies for Poly (ADP-ribose) polymerase (PARP), β-actin andcell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mMbeta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin), primaryantibodies against cleaved caspase 3 and cleaved caspase 9, Survivin,Bcl-X_(L) and secondary antibodies were obtained from commerciallyavailable supply.

Cell Viability Studies by MTS Assay

The PANC-1 and BxPC-3 cells (5×10³) were seeded and incubated overnightin 96-well culture plates. The medium was then removed and replaced witha fresh medium containing 20, 30, 40 or 50 μl of PJ (1500 mg/mL GE) perml of cell prior to 72 h of incubation. After that, 20 μl of MTS assayreagent was added to each well and incubated for 2 h at 37° C. in ahumidified, 5% CO₂ atmosphere. The reading was then recorded inabsorbance at 490 nm using plate reader. This assay is repeated byreplacing PJ with dimethyl sulfoxide (DMSO) as the vehicle control. Eachvariant of the experiment was performed in triplicate.

Histone/DNA ELISA for Detection of Apoptosis

The Cell Death Detection Enzyme-linked immunosorbent assay (ELISA) Kitwas used to detect apoptosis in PaCa cells. One million cells wereseeded and incubated overnight in six-well plates before treating withPJ or control for 72 h. The cells were then lysed and the cytoplasmichistone/DNA fragments were extracted and incubated in microtiter platemodules coated with anti-histone antibody. Peroxidase-conjugatedanti-DNA antibody were used to detect the immobilized histone/DNAfragment before color development with2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substratefor peroxidase. The spectrophotometric absorbance of the samples wasdetermined by using plate reader at wavelength of 405 nm.

Clonogenic Assay

One million cells were seeded and incubated overnight in a 100 mm dish.The cells were cultured with 20, 30, 40 or 50 μl of PJ or control for 72h prior to viable cell counting and plating with about 5,000 cells perplate. The cells were then incubated for 21 days at 37° C. in a 5% CO₂incubator. All the colonies were fixed in 4% paraformaldehyde andstained with 2% crystal violet.

Flow Cytometry and Cell Cycle Analysis

Cells were seeded and incubated overnight in 100 mm dish beforesubjecting all the cells for starvation for 24 h. The cells werereleased into control or PJ containing media for 72 h of incubation.Subsequently, cells were collected and fixed with ice-cold 70% (v/v)ethanol for 24 h. After subjecting cells for centrifugation at 3000×gfor 5 min, the cell pellet was washed with PBS (pH 7.4) and resuspendedin PBS containing propidium iodide (50 Hg/mL), and DNase-free RNase (1μg/mL). Samples were then incubated at room temperature for 2 h, and DNAcontent was determined using a flow cytometer.

Annexin V-FITC Method for Apoptosis Analysis

Annexin V-Fluorescein isothiocyanate (FITC) apoptosis detection kit wasused to measure the apoptotic cells. PANC-1 and BxPC-3 cells wereincubated in the presence or absence of PJ for 48 h before subjectingcells for trypsinisation, washing with ice-cold PBS and resuspension in1× binding buffer at a concentration of 10⁵ cells per ml in a totalvolume of 100 μl. The cells were then added with 51 μl of Annexin V-FITCand 5 μl of PI (Propidium Iodide) and were kept in the dark for 20 minat room temperature. Finally, each tube was added with 400 μl of 1×binding buffer and the number of apoptotic cells was analyzed using flowcytometer.

Wound Healing Assay

PANC-1 and BxPC-3 were seeded and incubated overnight in a six wellplate at a concentration of 4×10⁵ cells per well. After incubation,culture media were removed and a scratch wound across each well was madeusing fine tips. In order to ensure no loosely held cells were attachednear to wound areas, the wound areas were washed three times with PBS.Subsequently, the cells were cultured in presence or absence of PJ andthe wound images were taken as 0 h. After 20 h, wound healing pictureswere taken under microscope.

Cell Invasive Assay

A biocoat matrigel invasion kit was used to evaluate the tumor invasiveability according to the manufacturer's protocol. Around 5×10⁴ cells ofPANC-1 and BxPC-3 with basal media was transferred in each 6-well upperchamber in the presence or absence of PJ, while 3 ml of culture mediumwith 10% FBS was added into each lower chamber of 24-well plate. Thecells in the upper chamber were removed using a cotton stick after 20 hof incubation. Then the cells that invaded through the Matrigel matrixmembrane were stained with 4 μg/mL Calcein AM in Hanks buffered salineat 37° C. for 1 h. Each of experimental conditions was performed intriplicates. Subsequently, fluorescence of the invaded cells was readusing a microplate reader at excitation/emission wavelengths of 530/590nm. A fluorescent microscope was used to capture images of thesefluorescently labeled invasive cells.

Protein Extraction and Western Blotting

PANC-1 and BxPC-3 cell lines were treated with or without of PJ for 72 hto evaluate the effects of treatment on PARP, cleaved caspase 3, cleavedcaspase 9, Survivin, Bcl-X_(L), and β-actin expressions. Cells werelysed in cold lysis buffer for 30 mins on ice and the proteinconcentrations were determined using a protein assay kit. The sampleswere loaded on 10% SDS-polyacrylamide gels and subjected toelectrophoresis. After electrophoresis, the gel was electrophoreticallytransferred to a nitrocellulose membrane using transfer buffer (25 mMTris, 190 mM glycine, 20% methanol) in a transfer apparatus. Themembranes were incubated for 1 h at room temperature with 5% nonfatdried milk in Ix tris-buffered saline (TBS) buffer containing 0.1%polysorbate 20 (TBS-T) before incubating overnight at 4° C. with primaryantibodies. The membranes were subjected to washing for 3 times withTBS-T and incubated with the secondary antibodies containing 2% bovineserum albumin (BSA) for 2 h at room temperature before measuring signalintensity using a chemiluminescent imager.

Real-Time Quantitative PCR for Gene Expression Analysis

Total RNA was isolated according to the kit manufacturer's protocols.First strand cDNA synthesis was performed on 2 μg of total RNA from eachsample using TaqMan reverse transcription reagents kit in a total volumeof 20 μl. Reverse transcription reaction were performed at 25° C. for 10min, followed by 48° C. for 30 min and 95° C. for 5 min. Real-time PCRanalysis were performed and sequences of the primers sets used for thisanalysis are as follows: MMP-9, forward primer (5′-CGG AGT GAG TTG AACCAG-3′) and reverse primer (5′-GTC CCA GTG GGG ATT TAC-3′); VEGF,forward primer (5′-GCC TTG CCT TGC TGC TCT AC-3′) and reverse primer(5′-TTC TGC CCTCCT CCT TCT GC-3′); GAPDH, forward primer (5′-CAG TGA GCTTCC CGT TCAG-3′) and reverse primer (5′-ACC CAG AAG ACT GTG GAT GG-3′).

All these primers were verified by virtual PCR, and primerconcentrations were optimized to avoid primer dimer formation. Real-timePCR amplifications were performed using 2×SYBR Green PCR Master Mix. Twomicroliters of RT reaction were used for a total volume of 25 μlquantitative PCR reactions. The thermal profile for SYBR real-time PCRwas 95° C. 10 min followed by 50 cycles of 95° C. 15 s and 60° C. 1 m.Data were analyzed according to the comparative fold increase ordecrease in gene expression determined by quantitation of normalizedGlyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression in eachsample.

Microwell Colorimetric NF-κB Assay

In order to evaluate the binding activity of NF-κB, a transcriptionfactor ELISA kit for P65 was used according to the manufacturer'sprotocol. One million of PANC-1 and BxPC-3 cells were seeded andincubated overnight in 100 mm dish before treating with PJ or controlfor 72 h and nuclear protein extraction from each sample. Then, 2 μg ofeach sample were incubated in microplate coated with anti-p65 DNAsequence. Peroxidase-conjugated anti-DNA antibody was used to detect thep65-DNA binding complex before color development with ABTS substrate forperoxidase. The chemiluminescence and volume of the samples wererecorded and analyzed.

Data Analysis

Results are expressed as means±standard error of the mean (SEM) andstatistical comparisons between groups were done using one-way ANOVA.Values of p<0.05 were considered to be statistically significant andindividual p-values are reported in the figures.

Results Effects of PJ on Cell Growth/Survival of PaCa Cells

The cytotoxic potential of PJ on pancreatic cancer cell lines wasevaluated by treating PANC-1 and BxPC-3 cells with differentconcentrations of PJ and culture medium followed by the MTS andclonogenic assays as shown in FIG. 1. FIG. 1 shows the effect of PJ onpancreatic cancer cell line PANC-1 and BxPC-3 survival and growth (A andB) and clonogenicity (C and D).

Both cancer cell lines were treated with increasing concentrations ofPJ. Results are presented as mean±SEM of three assay replicates. Inreference to FIG. 1, * indicates P<0.05 and ** indicates P<0.01 versusrespective DMSO treated controls. The number of cells counted in thecontrol treatment was considered 100% and the number of cells in PJtreated cells was calculated in relative to this control.

In PANC-1 cell line, treatment with 20, 30, 40 and 50 μl/ml of PJ for 72h resulted in 35%, 51%, 75% and 91% of cell growth inhibition inrelative to control, respectively. Similarly, treatment on BxPC-3 cellline with 20, 30, 40 and 50 μl/ml of PJ for 72 h resulted in 12%, 30%,54% and 75% of cell growth inhibition in relative to controlrespectively. These results indicate that the efficiency of PJ as aninhibitor of pancreatic cancer cell growth.

Clonogenic assay as depicted in FIGS. 1C and 1D confirms the effects ofPJ on cell growth by revealing the treatment of PANC-1 and BxPC-3 withincreasing concentrations of PJ (30 and 40 μl/ml) resulted in a greaterreduction in the number of colonies, as indicated by reduced crystalviolet staining. As shown in FIG. 1, the results from the clonogenicassay were consistent with the MTS data where PJ significantly inhibitedpancreatic cancer cell growth. Summarizing both assays, it is found thatPJ inhibited cell proliferation and clonogenicity in both cell lineswhere PANC-1 cells were more sensitive to the effects of PJ than BxPC-3cells for all concentrations tested.

Induction of Apoptosis by PJ

FIG. 2 depicted the analysis of apoptotic ability of PJ with Histone/DNAELISA method (A and B) and flow cytometry (C and D). Cells were treatedwith increasing concentration of PJ or treated with DMSO as vehiclecontrol prior to incubation for 72 h. The incubation is followed bystaining with Annexin V and propidium iodide for flow cytometryanalysis. The flow cytometric cell distribution of cells is illustratedin dot plots (FIGS. 2C and 2D). The values represent ±SEM of triplicatesamples. * indicates P<0.05 and ** indicates P<0.01 versus DMSO-treatedcontrol groups.

FIGS. 2A and 2B show PJ induced apoptosis in PANC-1 (FIG. 2A) and BxPC-3(FIG. 2B) cell lines in a dose dependent manner. FIGS. 2C and 2Dindicates quantitation of apoptotic cells, as detected by Annexin Vstaining after treatment with 20 and 30 μl/ml of PJ, thus confirming theapoptosis-inducing effect of PJ in both cell lines. The result showedthat PJ treatment is statistically significant (**P<0.01) increasing inpercentage of apoptotic cells in both pancreatic cancer cell lines atIC₅₀ dosage of PJ.

Analysis of Cell Cycle Distribution after Treatment with PJ

The cell cycle distributions of PANC-1 and BxPC-3 cells treated withvarying concentrations of PJ were analyzed to determine possibleunderlying mechanism of cell proliferation suppression as depicted inFIG. 3A (PANC-1) and FIG. 3B (BxPC-3). The distribution of populationsin different cell cycle phases was quantified using flow cytometer after72 h of incubation.

FIGS. 3A and 3B indicate that both cell lines were arrested in the Sphase by PJ in a dose-depended manner. For PANC-1 cell (FIG. 3A), about28% cells are arrested in S phase in treatment group (40 μl/ml) ascompared to 25% in control cells. A similar observation was made inBxPC-3 cells (FIG. 3B) with 52% of cells arrested in S phase intreatment group (50 μl/ml) as compared to 21% in control cells.

Cell cycle distribution of cancer cells treated with increasingconcentrations of PJ are shown along with the percentages of the cellcycle stages, G0-G1, G2-M, and S phase. These findings reveal that PJinduced cell cycle arrest in the S phase for both cancer cell lines.

PJ Both Up-Regulates Pro-Apoptotic Genes and Down-RegulatesAnti-Apoptotic Genes

Western blotting represents the expression of PARP cleavage, caspases,Bcl-xL and Survivin in PANC-1 and BxPC-3 cancer cell lines after 72 h oftreatment with increasing PJ concentration to further elaborate themolecular mechanisms involve in PJ-induced apoptosis of pancreaticcancer cells. The β-actin protein was utilized as the protein loadingcontrol for this experiment.

Specifically, the expression of proteins involved in the induction ofcleaved caspase dependent apoptosis (caspase 3, caspase 9),anti-apoptotic Bcl-2 family (Bcl-xL) and anti-apoptotic protein—Survivinwere analyzed. FIG. 4 illustrates that PJ increases expression ofpro-apoptotic proteins, cleaved caspase 3 and 9 and PARP in both cancercell lines, while decreases production of anti-apoptotic proteins suchas Bcl-xL and Survivin in both PANC-1 and BxPC-3 cell lines.

This analysis demonstrates that PJ induces apoptosis in both cancer celllines through a dual-mechanistic approach: activation of apoptosisinducing caspases and inhibition of cell survival proteins.

Inhibition of NF-κB Activity with PJ

Microwell colorimetric NF-κB assay was performed to characterize theeffect of PJ on NF-κB activity. FIG. 5 shows the down regulation ofNF-κB activity as increasing concentration of PJ used in treatment onPANC-1 and BxPC-3 cells. FIGS. 5A and 5B shows the analysis of p65activity in PANC-1 and BxPC-3. P65 activation decreased in a similardose-dependent manner following PJ treatment in both cell lines. BothPANC-1 and BxPC-3 cell lines exhibited significantly lower NF-κB (p65)activity at higher concentrations of PJ used in treatment.

FIGS. 5C and 5D shows the effect of PJ on VEGF and MMP-9 expression inrelative to DMSO treated control. VEGF and MMP-9 expression wassignificantly reduced in treatment with PJ at dose-dependent manner. Theeffect on MMP-9 was more pronounced in both cancer cell lines asobserved in FIGS. 5C and 5D. Vascular endothelial growth factor (VEGF)and matrix metalloproteinase 9 (MMP9) being the downstream genes ofNF-κB, are responsible for cancer cell invasion and migrationexpression. The expressions of VEGF and MMP9 were measured by real timePCR after reverse transcriptase PCR. These results strongly suggest thatPJ inhibited NF-kB activity and its target genes expressions.

The results are presented as mean±SEM of three assay replicates. *indicates P<0.05 and ** indicates P<0.01 versus respective DMSO treatedcontrols.

Inhibition of Cell Migration and Invasion by PJ

The cell invasion and wound healing assays were used to analyze theeffect of PJ on invasion and migration on the pancreatic cancer celllines. FIG. 6 show PJ inhibits migration and cell invasion in PANC-1 andBxPC-1 cell lines in dose-dependent manner. Cells were cultured invarying concentrations of PJ for 20 h and were photographed afterstaining with Calcein AM. Cells cultured in the absence of PJ are usedas control. FIGS. 6A and 6B illustrates the fluorescence of the invadedcells in PANC-1 and BxPC-1 cell lines respectively. PJ decreased cellinvasion in a dose dependent manner, as semi-quantified by thefluorescence index reading. Results are presented as mean±SEM of threeassay replicates. * indicates P<0.05 and ** indicates P<0.01 versusrespective DMSO treated controls.

The wound healing assay shows the cell migration in PANC-1 and BxPC-1cell lines as depicted in FIGS. 6C and 6D respectively. When the cellsreached confluence, a single wound was scratched across each well. After20 h incubation with various concentrations of PJ, wound closure areaswere visualized using a microscope. These results support the cellinvasion findings that increasing PJ concentrations prevents cellmigration in an increased dose-dependent manner. Thus, this demonstratesthat PJ effectively retards migration and metastasis in PANC-1 andBxPC-3.

Present invention discloses PJ which inhibits proliferation, growth,migration, and invasion by inducing apoptosis, cell cycle arrest, anddown-regulating NF-κB activity in both cell lines.

This statement is supported by various experiments such as MTS andclonogenic assays wherein PJ inhibited cell proliferation andclonogenicity in both cell lines. Besides that, PJ was tested to beeffective in inducing apoptosis in a dose-dependent manner for PANC-1and BxPC-3. Furthermore, the flow cytometry analysis on cell cycledistribution revealed that PJ induces cell cycle arrest in the S phasefor both PANC-1 and BxPC-3 cells. Together, these data suggest PJinhibits cell growth, induces apoptosis and induces cell cycle arrest inthese cancer cell lines and hence could prove to be an effectiveantitumor agent.

Western blot analysis evaluated the expression of caspases and PARPprotein. The experiment data also demonstrated that PJ simultaneouslycaused a decrease in the expressions of the anti-apoptotic proteins suchas Bcl-xL and Survivin in both PANC-1 and BxPC-3, and also increase inthe expressions of pro-apoptotic proteins such as cleaved caspase 3,cleaved caspase 9 and cleaved PARP. Hence, present invention disclosesthat the apoptotic capacity of PJ is induced through a dual mechanism,demonstrating that PJ can indeed be a powerful nutraceutical againstcancer.

NF-κB plays important roles in many cellular processes including cellproliferation, anti-apoptosis invasion, and angiogenesis all of whichare crucial for cancer development and progression. By using thechemilluminance NF-κB ELISA kit, we demonstrated that P65 activitysignificantly decreased as PJ concentration increased in both cell linesas shown in FIGS. 5A and 5B. Since VEGF and MMP9 are known to beinvolved in downstream signaling of NF-κB responsible for cell migrationand invasion, relative expressions of VEGF and MMP9 for both cell lineswere evaluated. The study shows the expression of both VEGF and MMP9 wassignificantly decreased in response to increased PJ concentrations inboth PANC-1 and BxPC-3 cell lines as shown in FIGS. 5C and 5D.

The extracts from oil palm disclosed in present invention have alsoexhibited properties to inhibit cell invasion and migration. The currentstudies show that PJ caused a dose-dependent reduction in cell invasionand migration in both PANC-1 and BxPC-3 cells as shown in FIG. 6. Thisfurther supports PJ's candidacy in treating cancer.

Thus, present invention has disclosed the extracts from oil palm to workas a multi-faceted chemotherapeutic agent against pancreatic cancer.Benefits of its consumption may potentially involve inhibition of cellproliferation and growth, and induction of cell cycle arrest andapoptosis. The mechanisms through which these actions occur have alsobeen partially elucidated in this study, the most important of which isthe evident down-regulation of the NF-κB pathway.

The composition as disclosed in present invention may be provided ascompounds with pharmaceutically acceptable carriers. Present inventionfurther discloses the use of therapeutically effective amount of acomposition in the preparation of a medicament for preventing orinhibiting the growth of pancreatic cancer in an individual byadministering to an individual in need thereof. The composition isaccompanied with or without conventional chemotherapy or radiationtherapy or surgery, or it may be administered orally or as a foodsupplement.

It is understood by a person skilled in the art that the methods forexperiments and studies are described as exemplifications herein andthus the results are not intended, however, to limit or restrict thescope of the invention in any way and should not be construed asproviding conditions, parameters, agents, chemicals or startingmaterials which must be utilized exclusively in order to practice thepresent invention. It is therefore understood that the invention may bepracticed, within the scope of the appended claims, with equivalentmethods for the experiments than as specifically described and stated inclaims.

What is claimed is:
 1. A composition to inhibit pancreatic cancer cellproliferation and clonogenicity, wherein said composition comprisesplant extracts.
 2. The composition as claimed in claim 1, wherein saidplant extract is from oil palm.
 3. The composition as claimed in claim1, wherein said plant extracts is from vegetation liquor.
 4. Thecomposition as claimed in claim 3, wherein said vegetation liquorfurther comprising phenolics.
 5. The composition as claimed in claim 1,wherein said composition is useful for prevention of pancreatic cancerand diseases related thereto.
 6. The composition as claimed in claim 1,wherein said pancreatic cancer is from PANC-1 cell line.
 7. Thecomposition as claimed in claim 1, wherein said pancreatic cancer isfrom BxPC-3 cell line.
 8. The composition as claimed in claim 1, whereinsaid composition induces apoptosis.
 9. The composition as claimed inclaim 1, wherein said composition regulates gene expression.
 10. Thecomposition as claimed in claim 8, wherein said apoptosis is associatedwith inhibiting cell survival proteins.
 11. The composition as claimedin claim 8, wherein said apoptosis is associated with inhibitinganti-apoptotic protein.
 12. The composition as claimed in claim 11,wherein said anti-apoptotic protein is Survivin.
 13. The composition asclaimed in claim 9, wherein said gene expression regulated is associatedwith Bcl-2 family.
 14. The composition as claimed in claim 9, whereinsaid gene expression is associated with down-regulating Bcl-XLexpression.
 15. The composition as claimed in claim 8, wherein saidapoptosis is associated with increased expression of pro-apoptoticproteins.
 16. The composition as claimed in claim 15, wherein saidpro-apoptotic proteins are cleaved caspase 3, caspase 9 or Poly(ADP-ribose) polymerase (PARP).
 17. The composition as claimed in claim1, wherein said composition induces cell cycle arrest in S phase. 18.The composition as claimed in claim 1, wherein said composition inhibitsNF-κB activity.
 19. The composition as claimed in claim 18, wherein saidNF-κB activity inhibition is via down-regulating p65 subunit activity.20. The composition as claimed in claim 19, wherein said down-regulatingis associated with decreasing vascular endothelial growth factor (VEGF)gene expression.
 21. The composition as claimed in claim 19, whereinsaid down-regulating is associated with decreasing matrixmetalloproteinase 9 (MMP9) gene expression.
 22. The composition asclaimed in claim 1, wherein said composition reduces cell invasion. 23.The composition as claimed in claim 1, wherein said composition reducescell migration and metastasis.
 24. The composition as claimed in claim1, further comprising compounds with pharmaceutically acceptablecarriers.
 25. A method of using a therapeutically effective amount of acomposition to inhibit pancreatic cancer cell proliferation andclonogenicity, wherein said composition comprises plant extracts, andwherein the method is used in the preparation of a medicament forpreventing or inhibiting the growth of pancreatic cancer in anindividual by administering to an individual in need thereof.
 26. Themethod as claimed in claim 25, wherein the composition is accompaniedwith or without conventional chemotherapy or radiation therapy orsurgery,
 27. The method as claimed in claim 25, wherein the compositionis administered orally or as a food supplement.